This invention relates to a method of testing and is particularly though not exclusively concerned with a method of testing progesterone levels and pregnancy in cows.
Reproductive performance is one of the important factors determining the profitability of dairy herds. Ideally, the calving interval should average one year, but this can only be achieved if the pregnancy success and detection of oestrous rates are high and the interval between parturition and first service is less than 90 days (Bulma, D. C. and Lamming G. E. J Reprod. Fert (1978) 54: 447-58).
The basic physiology of pregnancy in cows is relatively well understood. Following ovulation, the release of an oocyte for fertilisation, which usually occurs 6-12 hours after the end of oestrous (occasionally up to 36 hours later), the rapidly growing corpus luteum (CL) is formed from follicular cells remaining in the ovary. One of the main functions of the CL is to secrete the steroid hormone progesterone (P4). During a normal 21-day oestrous cycle an animal shows measurable P4 levels between days 4-19 post oestrous and following the demise of the CL a period of 6 to 7 days with low P4 concentrations.
The early embryo is dependent on adequate progesterone concentrations. Fertilised ova remain in the oviduct until day 4 post-ovulation, and then migrate to the uterus. The embryo remains free or loosely attached until about day 25 of pregnancy. Prior to implantation of the embryo in the uterine wall, embryos are supported solely by secretions that accumulate in the uterine lumen. Changes in timing and magnitude of peripheral progesterone concentrations affect the uterine environment which in tun affects embryo viability. Progesterone is believed to play a major role in controlling the maternal secretion of nutrients, growth factors and enzymes required for successful embryo development. In sheep, changes in the content of total protein and of several metabolic enzymes in ovine uterine flushings follow the growth and regression of the CL (Ashworth et al (1989) Animal Reprod. Sci. 21: 213-221). Although in the cow, Day 16 post insemination is often regarded as the period for the material recognition of pregnancy it is well established that important physiological communications must have occurred between the embryo and its mother before that date. It has previously been shown that milk progesterone concentrations are higher in pregnant than in mated non-pregnant cows between days 10 and 18 of pregnancy (Lamming et al (1989) J. Reprod. and Ferth. Suppl. 37: 245-252).
Artificial insemination (A1) of cows is often used in dairy herds due to the ease of use and relative success of the process compared to natural insemination.
Them is currently major concern about the causes and impact of the increase in subfertility observed in dairy herds. Recent data derived from analysis of progesterone (P4) from both mild and close monitoring of a large number of cows in the UK, shows a decline in pregnancy rate to artificial insemination in untreated cows ranging from 65% in 1975 to 1982 compared to 44% in 1995 to 1999; a decline of approximately 1% per year (Darwash, A. O et al (1999a) Anim. Sci 68: 333-347; Royal M. D. et al (1999) Pro Br. Soc. Anim. Sci. (in press). Similar findings for the USA, using field records, have been reported all recently (Beam S. W. and W. R. Butler (1999). J. Reprod. Fertil. Suppl. 54 (in press). An increased rate of embryo loss is claimed to be a major component of the reduced reproductive efficiency. To quantify its extent, estimates show a fertilization rate of 89 to 100% (Bearden, H. J. et al (1956) J. Dairy Sci 39: 312-318; Diskin, M. G. and J. M. Sreenan (1980) J. Reprod. Fertil. 59: 463-468; Kidder, H. E. et as (1954) J. Dairy Sci. 37: 691-697) with the pregnancy rate falling to approximately 80% by Day 13 post insemination and to less than 60% by Day 19 to 20 post insemination (Peters, A. R. (1996). Anim. Breed Abstr. 64: 587-598; Screenan, J. M. and M. G. Diskin (1986) Embryo Mortality in Farm Animals, pp 1-11 Mamnus Nijhoff, Dordrecht). Consequently, early embryo loss by Day 16 (approximately 35%) and later embryo loss (approximately 10%) are of major concern. These losses have been observed particularly in cows showing atypical ovarian hormone patters before mating, which occurs in about 50% of animals (Lamming G. E. and A. O. Darwash (1998) Anim. Reprod. Sci. 52: 175-190). As a result, calving rates to AI in UK dairy herds are now generally less than 50% and in some herds below 40% (Darwash A. O. et al (1999a) Anim. Sci supra).
Currently, there is increasing interest in the importance of the magnitude and pattern of luteal P4 secretion post ovulation for both oestrous cycle control and the establishment and maintenance of pregnancy. Monitoring of milk P4 concentrations facilitates examination of the impact of luteal P4 patterns on pregnancy rates.
Fertility is often suggested as a result of the xe2x80x9ctimelyxe2x80x9d availability of P4 concentrations between days 4 to 10 post AI (Ahmad, N et al (1996) J. Anim. Sci. 74: 1943-1952; Albihin, A. H et al (1991) Anim. Reprod. Sci. 26: 193-210; Erb, R. E. et al (1976) Theriogenology 5: 227-242; Hansel, W. (1981) J. Reprod. and Fertil. Suppi. 30: 231-239; Lamming, G. E. et al (1989) J. Reprod and Fertil. Suppl. 37: 245-252; Lamming G. E and A. O Darwash (1995) Biol. of Reprod. 52: Suppl. 1, abstr 63; Larson, S. F. et al (1997) J. Dariy Sci. 80: 1288-1295; Maurer, R. R. and S. E. Echternkamp (1982) Theriogenology, 17: 11-22; Parkinson, T. J et al (1994) Theriogenology 41: 1115-1126). Using daily milk sampling, measurable concentrations of P4 (xe2x89xa73 ng/ml) were detected in individual cows on Days 4 to 6 post-oestus (Darwash, A. O et al (1998) Fertil. and Reprod. Grub, Germany, November 1997; Darwash, A. O et al (1999b) Anim Sci. (in press) which coincides with Days 3 to 4 or 5 to 6 post AI.
Although insuffcient circulating P4 concentration has often been suggested as the cause of early embryonic mortality which contributes to lowered fertility, P4 concentrations essential for embryo survival have hitherto been un-defined. The present inventors have previously shown that a delay of as little as one day in the availability of adequate progesterone concentration can induce a sub-optimal uterine environment which is deterimental to embryo survival and speculated that determining critical levels of P4 after insemination may assist in developing a non-pregnancy test possibly as early as day 7 post AI (Dash A. O and Lamming G. E. J. Animal Breeding (1998) 2: 41-43). The lack of a definition for the most appropriate timing and magnitude of P4 needed during early pregnancy, has tempted a practice of blanket P4 supplementation at various periods post AI, with inconsistent pregnancy results. A degree of improvement in fertility was achieved using progesterone releasing devices, (PRID), (Robinson N. A. et al (1989) J. Dairy Sci 72: 202-207) or controlled internal drug release (CIDR) (Macmillian, K. L and A. J Peterson (1993) Anim. Reprod. Sci. 33: 1-25) but others recorded no effect using CIDR (Van Cleeff, J. et al (1991) Theriogenology 36: 795-807). In all previous studies, P4 supplementation was carried out as a blanket treatment regardless of whether the cow actually required it or not. The inventors believe that the indiscriminate use of progesterone supplementation, in fact, is the reason for the discrepancies between the result of studes on P4 supplementation described above. Blanket treatment in some animals which already produce adequate P4 levels may result in higher than optimal P4 levels shortly after service which are known to have a detrimental influence on embryo survival (Farin P. W and Farin C. E. (1994) Biology of Reproduction 52: Suppl. Abstr 15, 23).
Consequently, there is a clear need to define the precise requirement for luteal P4 secretion afer insemination and the conditions under which P4 supplementation is appropriate and effective.
Post insemination milk P4 concentrations in relation to pregnancy rates were studied in postpartum Holstein Friesian cows. In a preliminary study, analysis of milk samples showed that one day delay in milk P4 rise  greater than 3 ng/ml (mean xc2x1se 5.15xc2x10.27 v 4.08xc2x10.16 days) was associated with pregnancy failure. Based on this result two further studies were completed involving P4 analysis of milk samples taken on Days 0, 5 and 24. In Study I mean milk P4 concentration on Day 0 was 1.96xc2x10.10 ng/ml (n=1451). Pregnancy rates were significantly correlated with Day 0 milk P4 concentrations (r=xe2x88x920.91) ranging between 51.3% at milk P4 less than 3 ng/ml to 11.3% at  greater than 5 ng/ml. Milk P4 concentration on Day 5 after insemination was 5.93xc2x10.10 ng/ml. Pregnancy rat was 31.1% in 256 animals (17.6%) with post insemination luteal P4 insufficiency (P4 concentrations  less than 3 ng/ml) compared with 52.4% for animals with 3 to 9 ng/ml and there was a highly significant effect of parity (10.2% incidence first lactation rising to 29.5% by third lactation). On Day 24, the mean miLk P4 concentration was 15.98xc2x10.32 ng/ml. Predicting pregnancy from Day 24 milk P4 concentrations was accurate above 16 ng/ml and varied between 27.3% in animals inseminated at an inappropriate time in relation to ovulation compared to 83.3% for animals with low Day 0 ( less than 3 ng/ml) and adequate P4 concentrations on Day 5 ( greater than 3 ng/ml).
In Study II P4 supplementation of cows with  less than 3 ng/ml P4 on Day 5 significantly increased pregnancy rates (from 28.9 to 57.9%) where pretreatment concentrations were between 1 and 2 ng/ml but not in animals above or below this range.
Data on milk P4 concentrations on the day of insemination and on Days 5 and 24 thereafter provides valuable information on the potential fertility status of cows. Milk P4 levels on Day 5 can be used to select suitable animals for P4 supplementation to improve pregnancy rates. Luteal P4 insufficiency on Day 5 may be used as an early test for non-pregnancy.
According to one aspect of the invention there is provided a method of predicting pregnancy in an inseminated cow, the method comprising taking a milk sample from the cow in which a milk progesterone concentration of  greater than 3 ng/ml by at least Day 4 post insemination is indicative that the animal is pregnant.
The method is non invasive and advantageous in that the milk samples can be readily obtained from the animal during the normal milking routine, being taken for example by the farmer.
According to another aspect of the invention, there is provided a method of improving pregnancy success in cows, the method comprising analysing milk progesterone levels from a cow and supplying the cow with progesterone, or a functional equivalent thereof if the milk progosterono level is  less than 3 ng/mlxe2x88x921, and where the progesterone levels are measured from at least one of Day 4, 5 and 6 post insemination.
Using this method, the likelihood of a successful pregnancy can be improved without the use of blanket administration of progesterone or suitable progestagen, i.e. a compound having equivalent effects, particularly biological effects, to progesterone. In this way, indiscriminate use of hormones can be avoided.
According to another aspect of the invention, there is provided a method assessing a cow for suitability for progesterone replacement therapy, comprising assessing milk progesterone levels in the cow at least one of Day 4, Day 5 or 6 post insemination. Similarly using this method, the indiscriminate use of hormones can be avoided.
Embryo transfer in cattle is being used to a limited extent having been used on 1% of the total UK milk cow population in 1997 (24,000 recipients in 2.5 million cows. However, it is relatively expensive to perform. It is desirable to improve the success rate of embryo transfer.
According to another aspect of the invention, there is provided a method of embryo transfer in cows, the method comprising selecting as recipients for embyro transfer cows on the basis of milk progesterone levels taken on at least one of Day 4 to 6 post oestrous.